Synthesis of N-substituted oligomers

ABSTRACT

A solid-phase method for the synthesis of N-substituted oligomers, such as poly (N-substituted glycines) (referred to herein as poly NSGs) is used to obtain oligomers, such as poly NSGs of potential therapeutic interest which poly NSGs can have a wide variety of side-chain substituents. Each N-substituted glycine monomer is assembled from two &#34;sub-monomers&#34; directly on the solid support. Each cycle of monomer addition consists of two steps: (1) acylation of a secondary amine bound to the support with an acylating agent comprising a leaving group capable of nucleophilic is displacement by --NH 2 , such as a haloacetic acid, and (2) introduction of the side-chain by nucleophilic displacement of the leaving group, such as halogen (as a resin-bound α-haloacetamide) with a sufficient amount of a second sub-monomer comprising an --NH 2  group, such as a primary amine, alkoxyamine, semicarbazide, acyl hydrazide, carbazate or the like. Repetition of the two step cycle of acylation and displacement gives the desired oligomers. The efficient synthesis of a wide variety of oligomeric NSGs using automated synthesis technology of the present method makes these oligomers attractive candidates for the generation and rapid screening of diverse peptidomimetic libraries. The oligomers of the invention, such as N-substituted glycines (i.e. poly NSGs) disclosed here provide a new class of peptide-like compounds not found in nature, but which are synthetically accessible and have been shown to possess significant biological activity and proteolytic stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional, of application Ser. No. 08/277,228,filed 18 Jul. 1994, now abandoned, which is a continuation-in-part ofour earlier filed U.S. application Ser. No. 08/126,539 filed Sep. 24,1993, now abandoned, which application is a continuation-in-part of ourearlier filed U.S. patent application Ser. No. 07/950,853 filed Sep. 24,1992, now abandoned, which applications are incorporated herein byreference in their entirety and to which applications we claim priorityunder 35 USC § 120.

FIELD OF THE INVENTION

The present invention relates generally to chemical synthesistechnologies. More particularly, the present invention relates to thesynthesis of N-substituted oligomers and particularly to peptide-likecompounds in the form of poly (N-substituted glycines) (referred toherein as poly NSGs) using solid-phase synthesis methods.

BACKGROUND OF THE INVENTION

Standard methods analogous to classical solid-phase methods for peptidesynthesis could be applied for the synthesis of NSGs. In accordance withsuch methods, the carboxylate of N,α-Fmoc-protected (and side-chainprotected) NSGs would be activated and then coupled to a resin-boundamino group. The Fmoc group is then removed followed by addition of thenext monomer. Thus, oligomeric NSGs could be prepared as condensationhomopolymers of N-substituted glycine. Such an approach is not desirabledue to the time and cost of preparing suitable quantities of a diverseset of protected N-substituted glycine monomers. Adding and removing theFmoc or other protective groups is time consuming and inefficient.

SUMMARY OF THE INVENTION

A synthesis method is disclosed whereby each N-substituted monomer isassembled from two "sub-monomers" directly on a solid substrate. Byvarying the basic structure and the substituents on the sub-monomers awide range of different oligomers can be produced, some of which mimicthe structure and activity of natural proteins and nucleic acids orportions thereof.

N-substituted oligomers, such as N-substituted glycines (poly NSGs) arecomprised of monomers prepared from two sub-monomers, the firstsub-monomer being an acylating agent comprising a leaving group capableof nucleophilic displacement, such as a haloacetic acid and a secondsub-monomer comprising a --NH₂ group, such as a primary amine. Thedirection of polymer synthesis with the sub-monomers occurs in thecarboxy to amino direction.

The solid-phase assembly of each monomer--and concurrent polymerformation--eliminates the need for N,α-protected monomers. Only reactiveside-chain functionalities need be protected.

Moreover, each sub-monomer is simpler in structure than the monomerspreviously used in synthesis of oligomers of amides, including aminoacids. Many of the sub-monomers are commercially available, whichdramatically reduces the time and cost required for poly NSG synthesis.

A primary object of the present invention is to provide a method ofsynthesizing poly (N-substituted amides) directly on a solid substratesupport.

Another object of the invention is to provide solid-phase methods forsynthesizing N-substituted oligomers, such as polymers of N-substitutedglycines, which oligomers can have a wide variety of side-chainsubstituents.

An advantage of the present invention is that the methods can be carriedout more efficiently than previous conventional synthesis usingsolid-phase methods.

An important embodiment of the invention is an automated and highlyefficient solid-phase method for is synthesizing a specific type ofoligomer which is referred to herein as poly N-substituted amides,particularly poly (N-substituted glycines).

Another advantage of the present invention is that the methodseliminates the need for N,α-protected monomers.

A feature of the present invention is that only the reactive side-chaingroups need be protected or blocked during the synthesis.

Yet another advantage of the present invention is that each sub-monomerof the monomer (and the oligomer) has a simple structure allowing forquick and efficient synthesis.

Another feature of the present invention is that many of the sub-monomercomponents used in connection with the invention are commerciallyavailable.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis and usage as more fullyset forth below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the results of a competitive binding assayused to determine the IC₅₀ values (and relative binding affinities) offour separate pools.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present solid-phase synthesis methodology is disclosed anddescribed, it is to be understood that the method of this invention isnot limited to the particular monomer or oligomer which can be preparedor to the conditions and techniques described as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms "a," "and," and "the" include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to "a reactant" such as "a sub-monomer" include aplurality and/or mixture of such monomers, reference to "anN,α-protected monomer" includes a plurality of such monomers andreference to "the polymer" includes a plurality and mixtures of suchpolymers and so forth.

All publications mentioned herein are incorporated herein by referencefor the purpose of disclosing and describing features of the inventionfor which the publications are cited in connection with.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

A number of terms are defined and used throughout the specification withthe following definitions provided for convenience.

Oligomer

The term "oligomer" includes polymers such as poly NSGs, produced by theprocess of the invention, including homopolymers, copolymers andinterpolymers of any length. More specifically, oligomers may becomprised of a single repeating monomer, two alternating monomer units,two or more monomer units randomly and/or deliberately spaced relativeto each other. Regardless of the type of poly amide produced, the polyamide of the invention is produced by the same general procedure whichincludes repeating a two-step cycle (described below in detail) whereina new monomer unit is added in each cycle until an oligomer of desiredlength is obtained. The oligomer is preferably 2-100 monomers, morepreferably 2-50, or 2-20, and most preferably 2-6 monomers.

Acyl Submonomer

The term "acyl submonomer" refers to an acylating reagent used in themethod of the invention. Acyl submonomers comprise a reactive carbonylor carbonyl equivalent, and a leaving group which may be displaced in anucleophilic displacement by an amine. "Carbonyl or carbonyl equivalent"includes, without limitation, carboxylic acids, esters, amides,anhydrides, acyl halides, and isocyantes (in the synthesis ofpolycarbamates of the invention). Esters and amides used will generallybe "reactive" forms., e.g., DIC adducts and the like. The acylsubmonomer may further comprise a side chain. Suitable acyl submonomersinclude, without limitation, bromoacetic acid, 3-bromopropionic acid,2-bromopropionic acid, 2-bromoethylisocyanate,2-bromoethylchloroformate, 6-phenyl-3-bromohexanoic acid,4-bromomethyl-benzoic acid, 4-bromomethyl-2-methoxybenzoic acid,5-bromomethyl-pyridine-2-carboxylic acid, and the like.

Amino Submonomer

The term "amino submonomer" refers to a compound containing an aminogroup capable of effecting a nucleophilic displacement of the leavinggroup in an acyl submonomer. The amino group may be primary, secondary,or tertiary. Addition of tertiary amines results in quarternary ammoniumsalts, and are preferably used as chain terminators (i.e., no furtheracylation of the oligomer is possible). Presently preferred aminosubmonomers are primary amines and hydrazides, although amides,carbamates, ureas, carbazides, carbazates, semicarbazides, and the likeare also suitable.

Sidechain

The term "sidechain" refers to a group attached to the polyamidebackbone of a compound of the invention, at either a nitrogen or carbonatom. Sidechains may be H, hydroxy, R_(a), --OR_(a), --NR_(a) R_(b),--SO₁,2,3,4 R_(a), --C(O)R_(a), --C(O)OR_(a), --OC(O)R_(a),--OC(O)OR_(a), --NR_(b) C(O)R_(a), --C(O)NR_(a) R_(b), --OC(O)NR_(a)R_(b), --NR_(c) C(O)NR_(a) R_(b), --NR_(b) C(O)OR_(a), --R_(a)--O--R_(b), --R_(a) --NR_(b) R_(c), --R_(a) --S--R_(b), --R_(a)--S(O)--R_(b), --R_(a) --S(O)₂ --R_(b), --OR_(a) --O--R_(b), --NR_(a)R_(b) --O--R_(c), --SO₁,2,3,4, R_(a) --O--R_(b), --C(O)R_(a) --O--R_(b),--C(O)OR_(a) --O--R_(b), --OC(O)R_(a) --O--R_(b), --OC(O)OR_(a)--O--R_(b), --NR_(b) C(O)R_(a) --O--R_(c), --C(O)NR_(a) R_(b)--O--R_(c), --OC(O)NR_(a) R_(b) --O--R_(c), --NR_(c) C(O)NR_(a) R_(b)--O--R_(d), --NR_(b) C(O)OR_(a) --O--R_(c), --OR_(a) --S--R_(b),--NR_(a) R_(b) --S--R_(c), --SO₁,2,3,4 R_(a) --S--R_(b), --C(O)R_(a)--S--R_(b), --C(O)OR_(a) --S--R_(b), --OC(O)R_(a) --S--R_(b),--OC(O)OR_(a) --S--R_(b), --NR_(b) C(O)R_(a) --S--R_(c), --C(O)NR_(a)R_(b) --S--R_(c), --OC(O)NR_(a) R_(b) --S--R_(c), --NR_(c) C(O)NR_(a)R_(b) --S--R_(d), --NR_(b) C(O)OR_(a) --S--R_(c), --OR_(a) --NR_(b)R_(d), --NR_(a) R_(b) --NR_(c) R_(d), --SO₁,2,3,4 R_(a) --NR_(b) R_(d),--C(O)R_(a) --NR_(b) R_(d), --C(O)OR_(a) --NR_(b) R_(d), --OC(O)R_(a)--N--R_(b) R_(d), --OC(O)OR_(a) --NR_(b) R_(d), --NR_(b) C(O)R_(a)--NR_(c) R_(d), --C(O)NR_(a) R_(b) --NR_(c) R_(d), --OC(O)NR_(a) R_(b)--NR_(c) R_(d), --NR_(c) C(O)NR_(a) R_(b) --NHR_(d), --NR_(b) C(O)OR_(a)--NR_(c) R_(d) ; where R_(a), R_(b), R_(c), and R_(d) are eachindependently alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl oraralkynyl;

where R_(a), R_(b), R_(c) and R_(d) are each substituted with 0-6 halo,NO₂, --OH, lower alkyl, --SH, --SO₃, --NH₂, lower acyl, lower acyloxy,lower alkylamino, lower dialkylamino, trihalomethyl, --CN, loweralkylthio, lower alkylsufinyl, or lower alkylsulfonyl.

Poly Amides

The term "poly amide" is used herein to describe oligomers of theinvention as described above, which oligomers are not restricted to poly(N-substituted glycines) as described below. The poly amide compounds ofthe invention are produced by repeating the two-step cycle which isshown within Reaction Scheme I. When the substituents on the nitrogenatom are always hydrogen, the resulting polymer is a poly (N-substitutedglycine), whereas when the substituent is a moiety other than hydrogen,the resulting compound is a poly amide. Poly amide includes polycarbamates as further described herein.

Poly (N-substituted glycines)

The terms poly (N-substituted glycines), oligo (N-substituted) glycines,and poly NSGs are used interchangeably herein and are produced using themethodology of the present invention. Poly NSGs are not peptides, i.e.,they are not composed of naturally-occurring amino acids linked inpeptide bonds. However, they may be designed so as to have structuralfeatures (e.g., reactive sites) which are closely related to naturallyoccurring peptides and proteins, and as such are useful as potentialtherapeutic agents and/or as binding sites on assays. The poly NSGsdisclosed herein can be designed so as to have a wide variety ofside-chain substituents--including substituents normally found onnatural amino acids and others not naturally occurring. For example, theinvention makes it possible to synthesize compounds having side chainswhich resemble pharmacophores of known drugs, e.g., phenoxyphenyl,2-adamantyl, and the like.

Sub-monomer

The term "sub-monomer" refers to an organic reactant used in the methodof the invention which is added to the substrate-bound material in astep of the invention. An "acyl sub-monomer" of the invention (the firstsub-monomer of scheme IA) is an acylating agent comprising a leavinggroup capable of nucleophilic displacement by any amino group, e.g.,--NH₂, --NRH or --NR₂. An "amino sub-monomer" (second sub-monomer ofscheme IA) is a displacing agent reactant comprising an --NH₂ group. Twosub-monomers react to form a monomer unit in a cycle of the invention,and repeating the cycle allows for the production of poly NSGs. Detailsof sub-monomer synthesis are described herein, in our parent U.S.application Ser. No. 07/950,853 now abandoned, and in our publication R.Zuckermann et al., J. Am. Chem. Soc. (1992) 114:10646-7, all of whichare incorporated herein by reference.

Molecular moiety

The term "molecular moiety" encompasses any atom or group of atomsattachable to a nitrogen atom or a carbon atom of the main-oligomerchain, thereby forming a side-chain off of the main chain of theoligomer, e.g., in CH₃ (R¹)NC(O)CH(R²)CH³, in which R¹ is a molecularmoiety attachable to the nitrogen atom of the oligomer main-chain,thereby forming a side-chain attached to the nitrogen atom, and R₂ is amolecular moiety attachable to the carbon atom of the oligomermain-chain, thereby forming a side-chain attached to the carbon atom.Thus, it is readily apparent to those of skill in the art of polypeptideor polyamide synthesis that a wide variety of molecular moieties can beused, including but not limited to hydrogen, and hydrocarbyl moietiessuch as alkyl, aryl and arylalkyl moieties. In the novel poly(N-substituted glycine) of Formula I below, at least one of themolecular moieties attachable to nitrogen is other than H (i.e. forms aside-chain substituted on the nitrogen).

Hydrocarbon, hydrocarbyl, hydrocarbylene

"Hydrocarbon" describes a compound, whereas "hydrocarbyl" and"hydrocarbylene" describe radicals with one or two hydrogens removedrespectively. Each are composed entirely of hydrogen and carbon atoms,and may be saturated or unsaturated, aliphatic, alicyclic or aromatic.When rings are included the structure usually includes one, two or threerings, which rings may be fused or bridged or spiro-fused.

Substituent, substituted and derivative

Substituent describes an atom or radical which is part of a firstmolecule in that it replaces another atom or radical of the firstmolecule. When a molecule is substituted, it is a derivative of amolecule bearing one or more substituents. Useful substituents in any ofthe sub-monomers of the invention include halo, alkyl, alkoxy,alkylthio, haloalkyl, haloalkoxy, halothio, disubstituted amino, and thelike, which replace an atom such as hydrogen attached to a nitrogen orcarbon.

Purine or pyrimidine base

A "purine or pyrimidine base" includes the natural nucleoside bases,such as A, T, G, C or U, and also derivatives thereof including thosepurines and pyrimidines substituted by one or more of alkyl,caboxyalkyl, amino, hydroxyl, halogen (i.e. fluoro, chloro, bromo, oriodo), thiol, or alkylthiol wherein the alkyl group contains from 1 toabout 6 carbon atoms. Non-limiting examples of purines and pyrimidinesinclude 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine,8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-hydroxyguanine,8-methylguanine, 8-thioguanine, 2-aminopurine, 5-ethylcytosine,5-methylcyosine, 5-bromouracil, 5-ethyluracil, 5-iodouracil,5-propyluracil, 2-methyladenine, methylthioadenine,N,N-diemethyladenine, 8-bromoadenine, 8-hydroxyadenine,6-hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl/cytosine) and thelike.

Leaving group

"Leaving group" means a moiety capable of nucleophilic displacement byan amine, e.g., --NH₂. Any leaving group can be used here provided it isreadily removed by nucleophilic displacement. Non-limiting examples ofleaving groups useful in the invention include halo, such as bromo,chloro, iodo, O-tosyl, O-triflyl, O-mesyl and the like.

Substrate

A "substrate" or solid support is a conventional solid support materialused in peptide synthesis. Non-limiting examples of such substrates orsupports include a variety of support resins and connectors to thesupport resins such as those which are photocleavable, DKP-forminglinkers (DKP is diketopiperazine; see, e.g., W090\09395 incorporatedherein by reference), TFA cleavable, HF cleavable, fluoride ioncleavable, reductively cleavable and base-labile linkers.

Protecting group

"Protecting group" means any group capable of preventing the atom towhich it is attached, usually oxygen or nitrogen, from participating inan undesired reaction or bonding, usually in a synthesis reaction.Protecting groups are also known to prevent reaction or bonding ofcarboxylic acids, thiols, and the like. Such groups and theirpreparation and introduction are conventional in the art and includesalts, esters and the like.

Method for Synthesis of Monomers from Sub-monomers

In the basic method of the invention, each N-substituted monomer issynthesized directly on a solid substrate (support) from two reactantswhich are referred to herein as sub-monomers.

Each monomer is produced by a synthesis cycle comprising two steps whichsteps may be carried out in either order as shown in Scheme IA andScheme IB. In accordance with Scheme IA, the first step comprisesacylation of a substrate-bound amine. In Scheme IA "P" is the solidphase substrate. The acylation of the amine on the subtrate is carriedout using a first sub-monomer acylating agent comprising a leaving groupcapable of nucleophilic displacement of a hydrogen of an amine e.g.,--NH₂, such as a haloacetic acid. The "OH" group of the acid reacts withthe "H" of the amine water is formed and the acid and amine are bound asshown in step IA. The second step of the monomer synthesis cyclecomprises the introduction of a side-chain by nucleophilic displacementof the leaving group, such as halogen or tosyl (represented by X), byproviding a sufficient amount, usually an excess, of a secondsub-monomer displacing agent comprising an amine, e.g., --NH₂ group,such as a primary amine. This is shown as step 2A within Reaction SchemeI.A.

Within Reaction Scheme I.B, the reaction begins with the acylating agentsub-monomer bound to the solid phase substrate represented by "P". Theleaving group "X" such as a halogen or tosyl extends from the surface ofthe substrate and is reacted, in the first step (step 1B), with anamine. At this point, an amine group now extends outward from thesubstrate, and is subjected (in step 2B) to acylation using asub-monomer acylating agent. This reaction is the same as per the step1A of Reaction Scheme I.A described above.

The basic two-step process of Reaction Scheme I (A or B) produces amonomer unit. In either Reaction Scheme, the steps 1 and 2 can berepeated any number of times to produce polymers (as per formula Vbelow) of any desired length as per monomers of structure I below. Thevariables shown in the structures can be changed to obtain a desiredresult. For example, by repeating step 1 and 2 five to twenty times andvarying the reactant it is possible to obtain a polymer with five totwenty monomer units with the units being different. Further, the basicsub-monomer structures can also be changed as below to obtain differentmonomer/polymer structures as in structures II, III and IV. ##STR1##

In each of the above, "P" is the solid phase surface, each R¹ and R³are, independently, any molecular moiety attached to a carbon atom, R²and R⁴ are, independently, any molecular moiety attached to a nitrogenatom, and n is an integer of from 1-10 (preferably 1 or 2). Any of R¹,R², R³ and R⁴ may include the twenty different side-chain moietiesattached to the twenty natural amino acids, i.e., --H of glycine; --CH₃of alanine; --CH(CH₃)₂ of valine; --CH₂ CH(CH₃)₂ of leucine;--CH(CH₃)CH₂ CH₃ of isoleucine; --CH₂ OH of serine; --CHOHCH₃ ofthreonine; --CH₂ SH of cysteine; --CH₂ CH₂ SCH₃ of methionine; --CH₂-(phenyl) of phenylalanine; --CH₂ -(phenyl)-OH of tyrosine; --CH₂-(indole group) of tryptophan; --CH₂ COO⁻ of aspartic acid; --CH₂ C(O)(NH₂) of aspargine; --CH₂ CH₂ COO⁻ of glutamic acid; --CH₂ CH₂ C(O)NH₂of glutamine; --CH₂ CH₂ CH₂ --N--(H)--C(NH₂)⁺ --NH₂ of arginine; --CH₂-(imidazole)⁺ group of histidine; and --CH₂ (CH₂)₃ NH₃ ⁺ of lysine.

Reaction Scheme I (A and B) includes some abbreviations which refer toreagents used in connection with the invention. For example, DMSO refersto dimethylsulfoxide, DIC refers to N,N-diisopropyl carbodiimide, andDMF refers to N,N-dimethylformamide.

Each step of the two-step method of the invention is usually conductedat about ambient temperature of 20° C. and pressure of 1 atmosphere.However, the reaction can also be carried out over a wide range oftemperatures between about 5° C. to about 80° C., and varies dependingon the solvent used. Depending on the temperature, the time of thetwo-step Reaction Scheme I can vary within the range of about 5 minutesto about 24 hours. The above temperature, times and reagents areapplicable to carrying out the reaction at atmospheric pressure. Otherpressures may be employed.

When the sub-monomers are liquids, each step can be conducted in theabsence of a solvent. However, an inert solvent is used when thesub-monomer is a solid or to facilitate the reaction. Suitable inertsolvents include ethers, such as dioxane, blocked amides, such asdimethyformamide, sulfoxides, such as dimethylsulfoxide, and the like.

The ratio of the reactants can vary. However, for highest yields it isdesirable to provide an excess of sub-monomer of from about 1.01 to 10times the amount of substrate-bound material, preferably, from about 1.5to 5 times the amount of substrate-bound material.

In the two-step cycle of the invention shown in Scheme I, the secondaryamine bound to the substrate is preferably an amine prepared from aprimary amine, and is bound (using conventional methodology) to asubstrate support base surface or solid phase (represented by the letter"P").

The first step of the cycle (in Scheme IA) is the acylation which iscarried out by reacting a first sub-monomer comprising an acylatingagent comprising a leaving group capable of nucleophilic displacement byan amine, e.g., --NH₂, such as a haloacetic acid, and especially abromoacetic acid representatively illustrated in Scheme I with thesubstrate-bound secondary amine to obtain an acylated amine.

The second step (in Scheme IA) of the two-step monomer synthesis methodof the invention is where the backbone nitrogen and side-chain or R²group of the monomer unit is added. In the second step (i.e. step 2A ofScheme IA), the acylated amine is reacted with a sufficient amount of asecond sub-monomer comprising an --NH₂ group, such as a primary amine orsecondary amine, alkoxyamine, semicarbazide, carbazate, acyl hydrazideor the like, which includes the R² group (i.e., the side-chain group),which is to be added at this monomer position in the oligomer. Thereaction of the second sub-monomer is preferably accomplished by addinga sufficient amount, usually an excess, of the second sub-monomer whichcauses a nucleophilic displacement of the leaving group, which isrepresentatively illustrated as the bromine shown in Scheme I.

Preparation of cyclic peptoids via the Sub-monomer Method

Cyclic peptoids have been prepared by the sub-monomer method. A generalReaction Scheme for such is shown below. ##STR2##

The key reaction to effect cyclization is the displacement of anN-terminal bromoacetamide with a side-chain nucleophile, generating a"head-to-side-chain" cyclic structure on the resin. The side-chainnucleophile is incorporated at the desired portion of the oligomer viastandard sub-monomer conditions. Typical nucleophiles are thiols andamines which can be protected. Preferred sub-monomers for this purposeare Moz-NH--CH₂ --CH₂ --NH₂, Alloc-NH--CH₂ --CH₂ --NH₂ and Trt-S--CH₂--CH₂ --NH₂. The oligomer is then elaborated until the desired lengthand is terminated with a bromoacetamide group. The side-chainnucleophile is then selectively deprotected and allowed by cyclize.

Specific examples of cyclic peptoid produced and the percentage yieldobtained are put forth below.

    __________________________________________________________________________    TRIMERS                                                                       R'"            R"            R'           MH.sup.+                                                                         Yield (%)                        __________________________________________________________________________     ##STR3##                                                                                     ##STR4##                                                                                    ##STR5##    536                                                                              15                                ##STR6##                                                                                     ##STR7##                                                                                    ##STR8##    575                                                                              29                                ##STR9##                                                                                     ##STR10##                                                                                   ##STR11##   600                                                                              25                                ##STR12##                                                                                    ##STR13##                                                                                   ##STR14##   705                                                                              20                                ##STR15##                                                                                    ##STR16##                                                                                   ##STR17##   870                                                                              45                               __________________________________________________________________________

Carbamate Synthesis via Submonomer Method

Each reaction step is followed by thorough washing with the reactionsolvents, and some combination of DCM, DMF, and/or MeOH. The reactionsare currently being optimized.

FMOC-protected Rink amide resin (250 mg, 0.43 mmole/g) was treated with20% piperidine/DMF for 20 minutes to remove N-terminal FMOC group. Afterthorough washing, the resin was acylated with FMOC-Sar-OH using standardmethods. The N-terminal FMOC group was removed with 20% piperidine/DMF.

The above resin was swollen with DCM and drained. A solution ofbromoethylchloroformate (180 μL, 1.67 mmol) and DIEA (260 μL, 1.5 mmol)in 5 mL of DCM was added to the resin and the resin was shaken for 45minutes. The resin was then washed well. To this resin was added asolution of butylamine (790 μL, 8.0 mmol) in DMF (3.2 mL) and it wasallowed to react for 2 hours with gentle shaking.

The acylation was repeated using bromoethylchloroformate (160 μL, 1.5mmol), and DIEA (260 μL, 1.5 mmol) in DCM (5 mL) for 45 minutes.Following washing, the resin was treated with a solution of benzylamine(875 μL, 8 mmol) in 3.2 mL DMF for 2 hours.

The resin was then washed and cleaved with 95% TFA/H₂ O for 90 minutes.The resulting solution was analyzed by C-4 RP-HPLC and MS. Three majorpeaks were obtained in approximately a 1:2:1 ratio. MS revealed themiddle peak to be the correct material, structure given. below. Theearly eluting peak was the deletion produce (incomplete reaction onBuNH₂ step) . The last peak appears to be the product from the secondacylation reaction, i.e., incomplete reaction on the final benzylaminestep. ##STR18##

The Butyl or Benzyl may be any side chain as defined above or beselected from the group consisting of halo, nitro, lower alkyl, lowercycloalkyl, --OH, --NR_(a) R_(b) where R_(a) and R_(b) are eachindependently --H or lower alkyl, --OR_(a), --C(O)R_(a), --OC(O)R_(a),--C(O)OR_(a), --OC(O)OR_(a), --(CH₂)_(n) --CX₁ X₂ X₃ where n is 0-6 andX₁₋₃ are each independently H or halo, --NC(O)R_(a), --C(O)NR_(a) R_(b),--OC(O)NR_(a) R_(b), or --NC(O)NR_(a) R_(b).

Photolithographic Method

The method of the invention may also be applied to theoptically-addressed spatial array technique described by Pirrung et al.,U.S. Pat. No. 5,143,854, incorporated herein by reference. Thistechnique uses analogs of semiconductor mask technology to formoligomeric compounds on the surface of any substrate in an array.Photolabile protecting groups are used to protect surface-boundcompounds from reaction. To add another monomer to a particular compound(i.e. a particular region in the array), one deprotects the compounds inthat region by illuminating or irradiating only that region. This isaccomplished using, e.g., a carefully aimed light source or laser, or amask which permits illumination only of the desired area(s). Usingsemiconductor-type photolithographic techniques, this method may bescaled down to extremely small sizes. Suitable photolabile protectinggroups include, without limitation, 6-nitroveratryloxycarbonyl (NVOC:3,4-dimethoxy-6-nitrobenzyloxycarbonyl), 2-nitrobenzyloxycarbonyl,α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDC), 5-bromo-7-nitroindolinyl,o-hydroxy-α-methylcinnamoyl, and 2-oxymethyleneanthraquinone.

The Pirrung et al. method is adapted to the method of the invention byusing photolabile protecting groups to protect oligomers ending in aminosub-monomers, synthesized in a spatially defined array. For example,acyl sub-monomers are coupled to a flat substrate in an array ofreaction zones (e.g., 8×12, 20×20, 100×100, etc.). A first aminosub-monomer is then coupled to all acyl sub-monomers, and is thenprotected, e.g. with NVOC. Zones are selected for coupling the nextmonomer (acyl sub-monomer and amino sub-monomer), and the remainingzones masked to prevent reaction. The selected zones are deprotected byillumination or irradiation, and are then reacted with the next acylsub-monomer followed by the next amino sub-monomer. The terminal aminosub-monomer is then protected again with NVOC (unless it is to befurther modified in the next round of synthesis), and the zones selectedfor the next monomer to be coupled. This cycle is repeated until alloligomers have been synthesized. The compounds may then be cleaved fromthe support, or may be assayed in situ (typically by assaying ability tobind fluorescently-labeled antibodies or ligands).

Halomethylbenzoic acids

In one embodiment of the invention, the first sub-monomer is ahalogenated organic acid, such as bromoacetic acid, chloromethylbenzoicacid and the like. The sub-monomer synthesis can accommodate theincorporation of several different halo-acids (e.g., bromoacetic acidand chloromethylbenzoic acid) in the same polymer chain to generatehybrid backbones. Furthermore, other derivatized aromatic acids could beused as well.

Acyl hydrazides

Acyl hydrazides, carbazates, semicarbazides and related compounds of theformula ##STR19## wherein X is a bond, --O--, --N--, or a hydrocarbylenegroup, can be used instead of amines as the second sub-monomerdisplacing agent in the method of the invention.

Oligomers generated by the sub-monomer synthesis using acyl hydrazideswill have a hydrogen bond donor and an acceptor group displayed in eachside-chain. This may allow stabilization of secondary and tertiarystructural motifs.

Acyl hydrazides are readily prepared from carboxylic acids/esters andhydrazine: ##STR20## Similarly, carbazates and semicarbazides can beprepared from alcohols or amines, p-nitrophenyl chloroformate andhydrazine: ##STR21## In this way, hydrazine can be viewed as an "adaptermolecule" that can link oligo (N-substituted) polymer backbones withcarboxylic acids, alcohols and amines. Thus, the sub-monomer synthesiscan be expanded to include not only amine-based diversity, but alcoholand carboxylic acid diversity as well. A very large number of alcoholsand carboxylic acids are commercially available and others can bereadily produced by known techniques.

The displacing agent can have a wide range of nucleophilicity, sterichinderance, volatility, side-chain protecting groups (when present),solubility and the like.

Any conventional amine (e.g., primary amine) can be used that does notcontain groups that would otherwise interfere with the reaction steps.This includes amines that have groups that are in a protected form,which protection may be subsequently removed. Non-limiting examples ofpreferred amines include 4-(2-aminoethyl)morpholine, aniline,benzylamine, cyclopentylamine, N-Boc-1,6-diaminohexane, glycine-OtBu,hexylamine, 2-methoxyethylamine, methylamine, tyramine and the like.

In another embodiment of the invention, the second sub-monomer is anacyl hydrazide. A benefit of such sub-monomers can be to stabilize thesecondary and tertiary motifs by providing a hydrogen bond donor and anacceptor group in each side-chain. Acyl hydrazides are readily preparedfrom carboxylic acids and esters and hydrazine using conventionaltechniques.

Similarly, carbazates and semicarbazides can be prepared conventionally,for example from alcohols or amines, p-nitrophenyl chloroformate andhydrazine.

Method of Synthesizing Oligomers

The basic two-step method of Scheme I yields a monomer unit. Another andimportant embodiment of the present invention is directed to theoligomer synthesis method comprising repeating the two-step cycle ofacylation and displacement. A particularly preferred embodiment of theinvention is a method of producing oligomers, such as poly NSGs.

Steps 1 and 2 can be repeated any desired number of cycles to obtain thedesired number of monomer units. Within each of the steps of each cycle,the variables R¹ and R⁴ shown within Scheme I.A can be varied in orderto produce different side-chain moieties. The terminal N is shownconnected to R⁴ and H here. However, this is done to allow other cyclesto add monomer units. The actual terminal --N containing group can becapped by providing alkyl and/or acyl groups for R³ and/or R⁴, asdefined for the poly NSGs of Formula V below. The variables R² and R³can be changed in each step of each cycle in order to obtain anydesirable side-chain moieties and resulting oligomer. Accordingly, itcan be seen that both Reaction Scheme I.A and I.B can be carried out toproduce any desired oligomer with any desired side-chain groups and withany desired ending moiety.

Different R groups are correctly positioned in the molecule by using thecorrect second sub-monomer in step 2 of each cycle. The resulting polyNSG consists of the desired sequence of monomer units.

Producing Oligomer Mixtures

It is also possible to use the invention to produce mixtures of polyamides which mixtures have known amounts of each poly amide by reacting(in step 2) mixtures of second sub-monomers with the acylated amine ofstep 1. By knowing or calculating the reaction rate constant for thereaction of each second sub-monomer with the acylated amine, it ispossible to calculate the proportional amounts of each product poly NSGwhich results and precisely determine the composition of the resultingmixture of poly NSGs. Such methodology is described as regards producingmixtures of conventional peptides by reacting conventional amino acidsbased on reaction rate constants in U.S. Pat. No. 5,225,533 issued Jul.6, 1993.

Further, the methods of the present invention could be applied in othermethods such as that of Houghten, R. A., Proc Natl Acad Sci USA (1985)82:5131-5135, which teaches a modification of the Merrifield methodusing individual polyethylene bags. In the general Merrifield method,the C-terminal amino acid of the desired peptide is attached to a solidsupport, and the peptide chain is formed by sequentially adding aminoacid residues, thus extending the chain to the N-terminus. The additionsare carried out in sequential steps involving deprotection, attachmentof the next amino acid residue in protected form, deprotection of thepeptide, attachment of the next protected residue, and so forth.

In the Houghten method, individual polyethylene bags containingC-terminal amino acids bound to solid support can be mixed and matchedthrough the sequential attachment procedures so that, for example,twenty bags containing different C-terminal residues attached to thesupport can be simultaneously deprotected and treated with the sameprotected amino acid residue to be next attached, and then recovered andtreated uniformly or differently, as desired. The resulting product ofthis procedure is a series of polyethylene bags each containing adifferent peptide sequence. Although each bag contains many peptides,all of the peptides in any one bag are the same. The peptides in eachbag can then be recovered and individually tested, e.g. via biologicalassays.

The present invention can be used with other methods in order to producemixtures of poly NSGs which include predetermined amounts of thedifferent poly NSGs in the mixtures, including equal molar amounts ofeach poly NSG in the mixture. The method can be used such that each polyNSG is present in the mixture in an amount such that it can beretrieved-and analyzed. Such mixture of poly NSGs can be generated bysynthetic algorithms that involve splitting pools of resin beads intoequal portions, coupling a unique NSG to each portion and then mixingthe portions (c.f. Furka, A., et al. (1991) Int. J. Pep. Pro. Res.,37:487-493; Lam, K. et al. (1991) Nature, 354:82-84; Houghten, R. et al.(1991) Nature, 354:84-86; Zuckermann, R. et al. (1991) Patent Appl. PCTWO 91/17823; Zuckermann, R. et al. (1992) Proc. Natl. Acad. Sci.89:4505-4509, incorporated herein by reference).

The methods of the present invention can also be used in an alternativemethod devised by Geysen, H. M., et al., Proc Natl Acad Sci USA (1984)81:3998-4002. See U.S. Pat. Nos. 4,833,092, 5,194,392, W086/06487 andW086/00991. This method is a modification of the Merrifield systemwherein the C-terminal amino acid residues are bound to solid supportsin the form of polyethylene pins and the pins treated individually orcollectively in sequence to attach the remaining amino acid residues.Without removing the peptides from support, these peptides can thenefficiently be assessed effectively and individually for the desiredactivity, e.g. interaction with a given antibody or receptor. The Geysenprocedure results in considerable gains in efficiency of both thesynthesis and testing procedures, while nevertheless producingindividual different peptides. The peptides can also be cleaved from thepins and assayed in solution.

Automated Synthesis

The preparation of NSG oligomers by reacting sub-monomers can be adaptedto an automated synthesizer (see Zuckermann, R. N., Kerr, J. M., Siani,M. & Banville, S., Int. J. Peptide Protein Res. (1992), Vol. 40 pp.497-506 and U.S. Pat. No. 5,252,296). Each cycle of monomer addition (asis shown in Scheme I) comprising the two steps: (1) an acylation step,and (2) a displacement step; with the proviso that there is noN,α-deprotection step.

Acylation of a secondary amine can be difficult, especially whencoupling an acyl sub-monomer. Accordingly, the acylation can befacilitated by the use of the acylating agent in the presence of acarboxylate. activator, such as a carbodiimide, as a potent acylatingagent mixture. Accordingly, it can be desirable for the first step ofacylation of a substrate-bound secondary amine with a first sub-monomer,such as a haloacetic acid (Lindner, W., Robey, F. A., Int. J. PeptideProtein Res., 30, 794-800 (1987); Robey, F. A., Fields, R. L., Anal.Biochem., 177, 373-377 (1989); Wetzel, R., Halualani, R., Stults, J. T.,Quan, C., Bioconjugate Chem., 1, 114-122 (1990)); Fisther, E. Ber.Dtsch. Chem. Ges. (1904), 37:3062-3071 uses a suitable carboxylateactivation method. A carbodiimide, haloacetyl halide or other suitableactivator can also be used.

The second step in the two-step method of the invention introduces theside-chain by nucleophilic displacement of the leaving group, which isgenerally a halogen (as a substrate-bound α-haloacetamide) with anexcess of a second sub-monomer comprising an amino group, e.g., an--NH₂, --NRH, --NR₂ group. The efficiency of the displacement ismodulated by the choice of the leaving group, for example, in the casewhere the leaving group is a halo atom (e.g., I>Cl).

Protection of carboxyl, thiol, amino and other reactive groups on theside-chain is desirable to minimize undesired side reactions. However,the mild reactivity of some side-chain moieties toward displacement oracylation can allow their optimal use without protection (e.g., indole,imidazole, phenol).

Oligomers

By use of the novel method of the invention, as shown in Reaction SchemeI and described above, it is possible to produce a wide range ofoligomers of the Formula I: ##STR22## wherein R is a sidechain asdefined above;

Z is a bond, --O--, --NC(O)W-- in which W-- is a bond, --O--, or --N--;

Y is a hydrocarbylene group or Ar wherein Ar is selected from the groupconsisting of arylene, heteroarylene having 1-4 heteroatoms,cycloalkylene, cycloalkenylene, heterocycloalkylene having 1-4heteroatoms, where Ar has from 1 to 3 rings, and said rings are joinedby a bond or alkylene radical, or are fused, bridged, or spiro-fused. Armay be substituted with 1-6 substituents selected from the groupconsisting of halo, nitro, lower alkyl, lower cycloalkyl, --OH, --NR_(a)R_(b) where R_(a) and R_(b) are each independently --H or lower alkyl,--OR_(a), --C(O)R_(a), --OC(O)R_(a), --C(O)OR_(a), --OC(O)OR_(a),--(CH₂)_(n) --CX₁ X₂ X₃ where n is 0-6 and X₁₋₃ are each independently Hor halo, --NC(O)R_(a), --C(O)NR_(a) R_(b), --OC(O)NR_(a) R_(b), or--NC(O)NR_(a) R_(b) ; and

n is an integer of from 2 to 2000.

When chloromethylbenzoic acids are used in place of bromoacetic acid,the oligomer has the Formula II: ##STR23##

R and R¹ may be any moiety connectable to a nitrogen atom, but each ispreferably, independently, a hydrocarbyl containing 1 to 30 carbonatoms.

The preferred method of synthesizing this oligomer is to modify theacylation step 1 to also include an activating agent, such as a meta orparachloromethylbenzoic acid anhydride. Thus, about 0.6M solution ofp-chloromethylbenzoic acid is combined with a carboxylate activator,such as about 0.5 equivalents of diisopropylcarbodiimide, for about 30minutes at room temperature. The precipitate (diisopropylurea) is thenremoved by filtration to yield the acylation solution. Acylationreactions are then conducted as previously described. The preactivationstep is used due to the slower rate of activation of the benzoic acidmoiety as compared to the acetic acid moiety.

The N-substituted oligomers of the invention can be varied by changingone or both of the reactants on Reaction Scheme I. Specifically, thereaction can be carried out using acyl hydrazide, carbazate,semicarbazide or a related compound of the structure: ##STR24## whereinX is --O--, --N--, or a bond and R¹ is as defined above in ReactionScheme I. When such reactants are used in Reaction Scheme I, it resultsin N-substituted oligomers wherein the oligomers are represented byFormula III: ##STR25## wherein X is a bond, O, N, or a hydrocarbylenegroup;

Y is a hydrocarbylene group or an arylene; and

R¹ is as defined above in Reaction Scheme I.

Alkoxyamines

When the second sub-monomer used to synthesize oligomer is analkoxyamine, the oligomer can have the Formula IV ##STR26## wherein Y isa hydrocarbylene group, such as methylene, or --CH₂ C₆ H₄ -- and R¹ isas defined above.

When carrying out Reaction Scheme I with an alkoxyamine, the alkoxyamineis used in the displacement reaction (step 2) as a 1.0-2.0M solution inDMSO.

The novel polyamide structures differ from polypeptides in that theside-chains are substituted on the nitrogen rather than (or in additionto) the α-carbon. One embodiment of the invention is directed tocompounds having the Formula V ##STR27## wherein R¹ and R⁴ are eachindependently any moiety attachable to the nitrogen atom;

R² and R³ are each independently any moiety attachable to the carbonatom, including --H or an alkyl moiety containing 1 to 6 carbon atoms,and are preferably --CH₃ and more preferably --H;

X are each, independently --HNR⁵ wherein R⁵ is as R¹ and X is preferably--NH₂, --OH, H and a straight or branched chain alkyl (1-6 carbons) ortwo lower alkyls, or X is --OR⁶ wherein R⁶ is --H or a lower alkyl (1-6carbons);

m is an integer of from 1 to 2,000, preferably 2-100, more preferably2-12, and most preferably 3-8; and

n is an integer of from 1 to 10 and is preferably 1 or 2.

Non-limiting examples of useful moieties for R¹, R², R³ and R⁴ (inparticular for R⁴) include the side-chain moieties present on anaturally occurring amino acid, i.e., --H of glycine; --CH₃ of alanine;--CH(CH₃)₂ of valine; --CH₂ CH(CH₃)₂ of leucine; --CH(CH₃)CH₂ CH₃ ofisoleucine; --CH₂ OH of serine; --CHOHCH₃ of threonine; --CH₂ SH ofcysteine; --CH₂ CH₂ SCH₃ of methionine; --CH₂ -(phenyl) ofphenylalanine; --CH₂ -(phenyl)-OH of tyrosine; --CH₂ -(indole group) oftryptophan; --CH₂ COO⁻ of aspartic acid; --CH₂ C(O)(NH₂) of aspargine;--CH₂ CH₂ COO⁻ of glutamic acid; --CH₂ CH₂ C(O)NH₂ of glutamine; --CH₂CH₂ CH₂ --N--(H)--C(NH2)⁺ --NH₂ of arginine; --CH₂ -(imidazole)⁺ groupof histidine; and --CH₂ (CH₂)₃ NH₃ ⁺ of lysine. Other useful moietiesfor R¹ -R⁴ (and in particular R¹ and R³) include alkyls containing 1-6carbons (straight or branched chains); aryls, aralkyls, nucleoside basesand derivatives thereof, carbohydrates and lipids.

There are a number of well known modified forms of the common aminoacids such as O-phosphoserine; O-phosphothreonine; O-phosphotyrosine;N-formylmethionine and glycinamide and the side-chains of these modifiedamino acids are also readily used as the R group on the compounds ofFormulas V and VI.

Typical R-groups used include pharmacophores and natural amino acids andderivatives thereof. The resulting poly NSGs will be biologicallyactive, e.g., mimic or block the activity of a naturally occurringpeptide or non-peptide molecule which adheres to a natural receptorsite.

Some compounds and groups of compounds are also important aspects of theinvention. One preferred subclass is directed to compounds of Formula VI##STR28## wherein R⁹ is a heterocyclic capable of forming hydrogen bondsand base pairing with purine or pyrimidine bases, including a nucleosidebase such as A, T, G, C or U or derivative hereof;

R¹ is defined above and preferably is --H or an alkyl moiety containing1 to 6 carbons, more preferably --CH₃, most preferably --H;

m is an integer of from 1 to 5 and is preferably 2;

n is an integer of from 1 to 2,000; and

X is a bond, --O--, --NR-- or O═C--O--.

Utility

The individual oligomers and mixtures of oligomers of the Invention areuseful in a variety of ways similar to that of conventionalnitrogen-based oligomers, proteins, polyamides and polypeptide-likeoligomers, for example, they can have one or more properties in bindingto various moieties, including proteins, such as enzymes, receptors,antibodies and the like, nucleic acids, carbohydrates, lipids, they canreact with enzymes to form products, or have other properties such asantigenic compounds for vaccines or diagnostic reagents, including asprobes. The liquid oligomers of the invention can also find utility asfunctional fluids, including solvents, antifreeze, and the like. Solidoligomers of the invention can also find utility as additives forfoodstuffs, as supports for diagnostic and other technical materials incommercial and research applications. Compounds as per the aboveformulas can also be used as enzyme inhibitors and in connection withaffinity chromatography.

Compounds of Formula VI are useful in binding to DNA and RNA and as suchcan be used as probes and/or in antisense technology. Useful probes canbe produced by synthesizing compounds of Formula VI, wherein R⁹ is anucleoside base, m is 2 and further wherein the monomer units of thecompound have the purine or pyrimidine nucleoside bases positioned in apredetermined sequence designed so as to provide for hybridization ofthe polymer with an appropriate DNA or RNA target.

Compounds and mixtures of compounds produced by Reaction Scheme Iinclude those encompassed by Formula I, II, III, IV, V, VI and VII.These compounds or mixtures thereof will, as indicated above, bind to avariety of receptors. Accordingly, such compounds or mixtures thereofcan be bound to a support to provide useful assay devices.

Because the compounds of Formula VI are used as probes, it is preferableto attach a suitable label to the polymer. Suitable labels and the meansof their attachment are known to those skilled in the art and includeradioactive, fluorescent and enzyme labels and the like.

Polymers of Formula VI can also be used in antisense technology when theR⁹ is a purine or pyrimidine base and the sequence of bases in thepolymer is designed so as to hybridize to and interrupt thetranscription or translation of appropriate DNA and RNA molecules whichare known to be pathogenic. When used in connection with antisensetechnology, the R¹ moiety may be a lipid moiety to provide for deliveryof the compound into the cell and into the nucleus of the cell.

Although compounds related to compound of Formula VI are disclosed inNielsen, P. E., Exholm, M., Berg, R. H. et al. Science, 254 (1991) 1497,by using the synthesis methods of the present invention the R¹ group canvary to obtain novel compounds of Formula VI which have a variety ofdesirable characteristics, such as improved cell penetration with R¹ asa lipid moiety. "Lipid moiety" means a moiety containing long-chainaliphatic hydrocarbons and their derivatives. Functional groups on thechain (general terminal group) include carboxylic acids, alcohols,amines, amino alcohols, and aldehydes. The term also includes waxes,fats and derived compounds.

Further, the R¹ moiety can be used as a site-specific attachment pointfor a metal chelator, a nuclease, and the like.

Mixtures of the oligomers of the invention synthesized as describedabove are useful in that they can be screened to determine which, ifany, of the NSGs have a given biological activity, e.g., bind to aknown. receptor. Methods of using such mixtures are taught in U.S. Pat.No. 5,010,175 issued Apr. 23, 1991 incorporated herein by reference.

Diagnosis and Therapy

The invention includes a method of antisense treatment comprisingadministering to a mammalian (human) cell in vitro or in vivo apharmaceutical formulation comprising a pharmaceutically acceptableexcipient carrier having dispersed therein a therapeutically effectiveamount of a compound of the Formula VI: ##STR29## All of the variablesare defined above.

The invention also includes a composition for diagnosis or therapycomprising an effective amount of an oligomer of the invention and aphysiologically acceptable excipient or carrier.

Physiologically acceptable and pharmaceutically acceptable excipientsand carriers for use with peptide and polyamide type reagents are wellknown to those of skill in the art.

By "physiologically or pharmaceutically acceptable carrier" as usedherein is meant any substantially non-toxic carrier for administrationin which the oligomers will remain stable and bioavailable when used.For example, the oligomer can be dissolved in a liquid, dispersed oremulsified in a medium in a conventional manner to form a liquidpreparation or is mixed with a semi-solid (gel) or solid carrier to forma paste, ointment, cream, lotion or the like.

Suitable carriers include water, petroleum jelly (vaseline), petrolatum,mineral oil, vegetable oil, animal oil, organic and inorganic waxes,such as microcrystalline, paraffin and ozocerite wax, natural polymers,such as xanthanes, gelatin, cellulose, or gum arabic, syntheticpolymers, such as discussed below, alcohols, polyols, water and thelike. Preferably, because of its non-toxic properties, the carrier is awater miscible carrier composition that is substantially miscible inwater. Such water miscible carrier composition can include those madewith one or more ingredients set forth above but can also includesustained or delayed release carrier, including water containing, waterdispersable or water soluble compositions, such as liposomes,microsponges, microspheres or microcapsules, aqueous base ointments,water-in-oil or oil-in-water emulsions or gels.

In one embodiment of the invention, the carrier comprises a sustainedrelease or delayed release carrier. The carrier is any material capableof sustained or delayed release of the oligomer to provide a moreefficient administration resulting in one or more of less frequentand/or decreased dosage of the protein growth factor, ease of handling,and extended or delayed effects. The carrier is capable of releasing theoligomer when exposed to the environment of the area for diagnosis ortreatment or by diffusing or by release dependent on the degree ofloading of the oligomer to the carrier in order to obtain releases ofthe oligomer. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, or microcapsules of natural and syntheticpolymers and the like. Examples of suitable carriers for sustained ordelayed release in a moist environment include gelatin, gum arabic,xanthane polymers; by degree of loading include lignin polymers and thelike; by oily, fatty or waxy environment include thermoplastic orflexible thermoset resin or elastomer including thermoplastic resinssuch as polyvinyl halides, polyvinyl esters, polyvinylidene halides andhalogenated polyolefins, elastomers such as brasiliensis, polydienes,and halogenated natural and synthetic rubbers, and flexible thermosetresins such as polyurethanes, epoxy resins and the like.

Preferably, the sustained or delayed release carrier is a liposome,microsponge, microphere or gel.

The compositions of the invention are administered by any suitablemeans, including injection, transdermal, intraocular, transmucosal,bucal, intrapulmonary, and oral. While not required, it is desirablethat parenteral compositions maintain the oligomer at the desiredlocation for about 24 to 48 hours; thus, sustained release formulationscan be used, including injectable and implantable formulations.

If desired, one or more additional ingredients can be combined in thecarrier: such as a moisturizer, vitamins, emulsifier, dispersing agent,wetting agent, odor-modifying agent, gelling agents, stabilizer,propellant, antimicrobial agents, sunscreen, and the like. Those ofskill in the art of diagnostic pharmaceutical formulations can readilyselect the appropriate specific additional ingredients and amountsthereof. Suitable non-limiting examples of additional ingredientsinclude stearyl alcohol, isopropyl myristate, sorbitan monooleate,polyoxyethylene stearate, propylene glycol, water, alkali or alkalineearth lauryl sulfate, methylparaben, octyl dimethyl-p-amino benzoic acid(Padimate O), uric acid, reticulan, polymucosaccharides, hyaluronicacids, aloe vera, lecithin, polyoxyethylene sorbitan monooleate,tocopherol (Vitamin E) or the like.

Preferably the carrier is a pH balanced buffered aqueous solution forinjection. However, the preferred carrier will vary with the mode ofadministration.

The compositions for administration usually contain from about 0.0001%to about 90% by weight of the oligomer compared to the total weight ofthe composition, preferably from about 0.5% to about 20% by weight ofthe oligomer compared to the total composition, and especially fromabout 2% to about 20% by weight of the oligomer compared to the totalcomposition.

The effective amount of the oligomer used for therapy or diagnosis ofcourse can vary depending on one or more of factors such as the specificoligomer used, the age and weight of the patient, the type offormulation and carrier ingredients, frequency of use, the type oftherapy or diagnosis preformed and the like. It is a simple matter forthose of skill in the art to determine the precise amounts to use takinginto consideration these factors and the present specification.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the synthesis of the present invention and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.) but some experimental errors and deviationshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade and pressure is at or near atmospheric.

Oligomer syntheses were performed by an automated synthesizer(Zuckermann, R. N., Kerr, J. M., Siani, M. & Banville, S., Int. J.Peptide Protein Res. (1992), Vol. 40 pp. 497-506). The syntheses wereconducted with Rink amide polystyrene resin (Rink, H., TetrahedronLett., 28, 3787-3790 (1987)) (50 μmol, substitution level 0.45 mmol/g)to avoid diketopiperazine formation. However, a variety of conventionalpeptide synthesis resins known to those skilled in the art can be usedin place of the polystyrene.

Acylation reactions were performed by addition of bromoacetic acid (600μmol, 83 mg) in DMF (0.83 mL), followed by addition ofN,N'-diisopropylcarbodiimide activator (660 μmol, 103 μL) in DMF (170μL). Reaction mixtures were agitated at room temperature for 30 min.Each acylation was repeated once before continuing to the displacementstep.

Displacement reactions were performed by addition of primary amine (2.0mmol) as 2.5M solutions in dimethylsulfoxide (1.0 mL), followed byagitation for 2 hr at room temperature. Optimization of displacementreactions was performed by varying amine concentrations from 0.25M to2.5M.

The resulting oligomers were deprotected/cleaved by treatment of theoligomer-resin with 95% trifluoroacetic acid in water (10 mL) for 20 minat room temperature, followed by filtration and lyophilization.

EXAMPLES 1-8

Eight representative penta-NSGs were prepared by the sub-monomer methodfrom a variety of amines, including poorly nucleophilic,sterically-hindered and side-chain protected amines. All compounds weresuccessfully synthesized as established by mass spectrometry, withisolated crude yields between 52 and 90%, and purities generally greaterthan 85% by HPLC. The purity, yields and mass spectrometry data on thepentamers were obtained and are shown below in Table I.

                                      TABLE I                                     __________________________________________________________________________    Oligomer                 purity (%).sup.a                                                                    yield (%).sup.b                                                                    MH.sup.-c                                 __________________________________________________________________________     ##STR30##               >85   90   583.5                                      ##STR31##               >85   74   753.2                                      ##STR32##               >85   79   713.4                                      ##STR33##               >85   70   1204.1                                     ##STR34##               >85   83   683.3                                      ##STR35##               >85   83   503.3                                      ##STR36##               >60   52   1018.4                                     ##STR37##               >85   .sup. 63.sup.d                                                                     588.4                                      ##STR38##               >65   .sup. 86.sup.d                                                                     2850.9                                    __________________________________________________________________________     .sup.a Determined by HPLC.                                                    .sup.b Determined from dry weight.                                            .sup.c Liquidmatrix secondaryion mass spectrometry.                           .sup.d Made from BocNH(CH.sub.2).sub.3NH.sub.2.                          

Optimization of penta-NSG synthesis was performed using combinations ofchloro, bromo and iodoacetic acid with both aniline and cyclohexylamine.Bromoacetic acid and iodoacetic acid proved superior to chloroaceticacid in forming penta-(N-phenylglycine) (79%, 83% and <5% yields,respectively). All three haloacetyl compounds successfully gave thepenta-(N-cyclohexylglycine) oligomer in >75%. yield. However, inclusionof 0.6M N-hydroxybenzotriazole in the acylation reactions (Robey, F. A.,Harris, T. A., Hegaard, N. H. H., Nguyen, A. K., Batinic, D. ChimicaOggi 27-31 (1992)) yielded <5% of the penta-(N-cyclohexylglycine)polymer.

In further optimization studies, the molar concentration of primaryamine was varied from 0.25M (4.0 equiv.) to 2.5M (40 equiv.) forn-butylamine, cyclopropylamine and diphenylethylamine using bromoaceticacid. Pentamers were obtained in >80% yield with n-butylamine andcyclopropylamine concentrations ≧1.0M, and diphenylethylamineconcentrations ≧2.5M.

EXAMPLE 9

A 25 mer, (N-n-butylglycine)₄ (N-(3-aminopropyl)glycine)!₅, wassynthesized by the sub-monomer method, thereby demonstrating the utilityof this method for the preparation of longer oligomers. Analytical HPLCwas performed on a Rainin HPX system controller with a C4 reversed-phaseHPL- column (Vydac, 25 cm×4.6 mm) and a gradient elution (solvent A: H₂O /0.1% TFA and solvent B: CH₃ CN/0.1% TFA; 10%-75% B in 35 min). Massspectroscopy confirmed the identity of this compound (MH+=2850.9) whichwas obtained in 86% yield and 65% purity by HPLC.

The efficient synthesis of a wide variety of oligomeric NSGs usingautomated synthesis technology, as presented here, makes these polymersattractive candidates for the generation and rapid screening of diversepeptidomimetic libraries.

EXAMPLE 10

Resin-bound amine in dimethyformamide (DMF) and 200 μl ofdiisopropylcarbondimide was acylated twice with 800 μl of 0.6Mbromoacetic acid in DMF for 30 minutes at room temperature. The acylatedresin-bound amine was washed three times with 2 mL of DMF.

The acylated resin-bound amine was treated with 1 mL of a primary amineof Table II as a 1-2M solution in dimethyl sulfoxide (DMSO) for twohours at room temperature. The above steps were repeated to form apentamer. The desired pentamer product was washed three times with 2 mLof DMF, and subjected to reversed-phase HPLC using a standardacetonitrile gradient (0-60% in 30 minutes) to give the desired pentamerin greater than 85% purity.

                  TABLE II                                                        ______________________________________                                        Material              Notes                                                   ______________________________________                                        4-(2-aminoethyl)morpholine                                                                     50     g     tertiary amine                                  aniline          100    g     weak nucleophile                                benzylamine      100    g                                                     cyclopentylamine 50     g     α-branched amine                          N--Boc-1,6-diaminohexane (HCl)                                                                 20     g     soluble at 1.5M/DMSO                            Glycine-OtBu (HCl)                                                                             50     g     protecting group                                hexylamine       100    mL                                                    2-methoxyethylamine                                                                            50     mL                                                    methylamine (40% w/v                                                                           100    mL    use without dilution                            in water)                                                                     tyramine         50     g     soluble at 1M/DMSO                              bromoacetic acid 200    g                                                     ______________________________________                                    

All of the amine compounds listed were soluble in DMSO at 2M, exceptwhere otherwise noted. Tyramine was slow to dissolve, but gentle warmingin a hot water bath speeded up the process. There was no need to protectthe phenol functionality. Methylamine was quite volatile, but its highsolubility in water allowed its use as an aqueous solution (undilutedfrom the bottle). Aniline was the least nucleophilic amine, but it stillworked at a 2M concentration.

The hydrochloride salts were prepared by dissolving the compounds inDMSO and then adding a molar equivalent aqueous HCl. The saltprecipitate was then removed by centrifugation, and the supernatantdried over molecular sieves.

Peptoid oligomers with a Rink amide linker were cleaved as follows:

25-50 μmol of support-bound oligomer was reacted with 2-4 mL of 95trifluoroacetic acid/5% water for 20-30 min at room temperature; dilutewith an equal volume of water, lyophilized, redissolve in 3-6 mL glacialacetic acid and relyophilized. The oligomers were usually powders ratherthan oils.

EXAMPLE 11

The compounds described in Tables IV and V were synthesized as pentamersrepresented by Formula VIII: ##STR39## where R=the side-chain listed inTables IV and V. All oligomers were analyzed by reverse phase HPLC andcharacterized by LSIMS mass spectrometry.

All compounds were synthesized by the solid-phase sub-monomer method aspreviously described, but with the above modifications.

                                      TABLE IV                                    __________________________________________________________________________    Homopentamers with a toluic acid backbone                                     generated by the sub-monomer method                                           RNH.sub.2         Yield (%)       Purity (%)                                                                         MH.sup.+                               __________________________________________________________________________     ##STR40##        60              90   1247.7                                  ##STR41##        72              80   973.8                                   ##STR42##        78              85   963.8                                  __________________________________________________________________________     ##STR43##                                                                

                  TABLE V                                                         ______________________________________                                        Synthesis of Hydrazide-containing Polymers                                    by the Sub-monomer Method.sup.a                                                ##STR44##             (%)Yield                                                                             (%)Purity                                                                            MH.sup.+                                 ______________________________________                                         ##STR45##            86     90      687.3.sup.b                               ##STR46##            86     90      719.3                                     ##STR47##            75     80      603.2.sup.c                               ##STR48##            60     85      811.8                                     ##STR49##            78     90      839.3                                     ##STR50##            50     90                                                ##STR51##            88     90      841.4                                     ##STR52##            70     80      603.2.sup.c                              ______________________________________                                         .sup.a Synthesized as pentamers in the format BnXBnXBn, where Bn = Nbenzy     glycine.                                                                      .sup.b Synthesized as a homopentamer.                                         .sup.c Deprotects upon TFA cleavage to give the underivatized hydrazide. 

EXAMPLE 12

The method of the invention was used to synthesize pentamers in theformat Bn-X-Bn-X-Bn, where Bn is N-benzylglycine using an alkoxyamine asthe second sub-monomer substituted by --NH₂. When the alkoxyamine wasmethoxyamine the yield was 76% and the purity by HPLC was 90%. Whenphenylmethoxyamine was used as the alkoxyamine, the yield was 56% andthe purity by HPLC was 50%.

                  TABLE V                                                         ______________________________________                                        Synthesis of Alkoxyamine-containing                                           Polymers by the Sub-monomer Method.sup.a                                                     Yield (%)                                                                              Purity (%)                                                                             MH.sup.+                                     ______________________________________                                        CH.sub.3ONH.sub.2                                                                              76         90                                                 ##STR53##       56         50                                                ______________________________________                                         .sup.a Synthesized as pentamers in the format BnXBnXBn, where Bn = Nbenzy     glycine.                                                                 

EXAMPLE 13

Synthesis of Ligands for α₁ Adrenergic Receptors

A. General Synthesis of Compounds

Oligomer synthesis was performed on a Rink amide polystyrene resin (0.61mmol/g, 1% crosslinked, 100-200 mesh). N,N-Dimethylformamide (DMF),dimethylsulfoxide (DMSO), methylene chloride, glacial acetic acid andtrifluoroacetic acid (TFA) were obtained from commercial suppliers andused without further purification. Piperidine, bromoacetic acid,N,N-diisopropylcarbodiimide (DIC), phenethylamine, 4-aminobiphenyl,tyramine, and other reagents were obtained from Aldrich and used withoutfurther purification.

All reactions were performed at room temperature in a 2.0 L vesselequipped with a 10 cm coarse glass frit. Agitation of the resin-reagentslurry was performed at every step by rotary shaking at 200 rpm.Filtration of the resin-reagent slurry was achieved by the applicationof vacuum.

A 2.0 L vessel was charged with Rink amide resin (100 g, 0.061 mol). Theresin was briefly swelled in DMF (1.5 L) with gentle agitation anddrained. The 9-fluorenylmethoxycarbonyl (Fmoc) group was then removed bytreatment with 20% piperidine/DMF (1.7 L, 1×5 min, followed by 1×20min). The resin was then washed with DMF (6×1.7 L). The remainder of thecompound was synthesized by performing three cycles of acylation withbromoacetic acid and displacement with an amine.

General acylation conditions (0.061 mol resin) Resin-bound amines werebromoacetylated by in situ activation with DIC. To the oligomer-resinwas added a DMF solution of bromoacetic acid (0.67M, 900 mL) is followedby DIC (neat, 93 mL, 0.60 mol) . The reaction mixture was agitated for30 min at room temperature. The mixture was drained and the reaction wasrepeated once. The resin was washed with DMF (3×1.7 L).

General displacement conditions (0.61 mol)

Resin-bound bromoacetamides were displaced by the addition of the amineas a solution in DMSO (1-2M, 1.0 L). The reaction mixture was agitatedat room temperature for 2 hours. The reaction mixture was drained andthe resin was washed with DMF (3×1.7 L). Phenethylamine and4-aminobiphenyl were used at 2.0M concentration, while tyramine andphenethylhydrazine were used at 1.0M.

General Cleavage and Purification

After completion of the synthesis the resin was washed with CH₂ Cl.sub.₂(3×1.7 L) and air dried for 5 minutes. The full length trimer wascleaved from the resin (0.061 mol) by treatment with 95% TFA/5% water(1.5 L) at room temperature for 15 minutes. The resin was then washedwith 95% TFA/5% water (1×1.0 L) and CH₂ Cl₂ (1×1 L). The filtrates werepooled and the solvent removed by rotary evaporation. The residue wasdissolved in glacial acetic acid (150 mL) and lyophilized.

EXAMPLE 14 Synthesis of Nhtyr-Nbiph-Nhphe

The compound Nhtyr-Nbiph-Nhphe was synthesized as described in Example18 above, using phenethylamine as the first amine added, 4-aminobiphenylas the second amine added, and 4-hydroxyphenethylamine as the thirdamine added.

After completion of the synthesis the resin was washed with CH₂ Cl₂(3×1.7 L) and air dried for 5 minutes. The full length trimer wascleaved from the resin (0.061 mol) by treatment with 95% TFA/5% water(1.5 L) at room temperature for 15 minutes. The resin was then washedwith 95% TFA/5% water (1×1.0 L) and CH₂ Cl₂ (1×1 L). The filtrates werepooled and the solvent removed by rotary evaporation. The residue wasdissolved in glacial acetic acid (150 mL) and lyophilized to afford alight yellow powder (1.7 g, 82% yield). The purity of the crude productwas determined to be 90% by reverse-phase HPLC. The product wascharacterized by FAB-mass spectrometry (MH⁺ =565).

EXAMPLE 15

Synthesis of Nhtyr-Npop-Nhphe

The compound Nhtyr-Npop-Nhphe was synthesized. as described in Example18 above, using phenethylamine as the first amine added,4-amino-1-phenoxybenzene as the second amine added, and4-hydroxyphenethylamine as the third amine added.

EXAMPLE 16 Synthesis of Backbone Variants

Proceeding as described in Example 18 above, but substituting3-bromopropanoic acid and 2-bromopropanoic acid for bromoacetic acid atsome positions, the following compounds were prepared:

Nhtyr-Nbiph-Nmhphe;

Nhtyr-Nbiph-Nphphe;

Nphtyr-Nbiph-Nhphe; and

Nhtyr-Npbiph-Nhphe.

EXAMPLE 17 Synthesis of Additional Compounds

Proceeding as described in Example 18 above, but substitutingphenethylamine, phenethylhydrazine and 3,4-methylenedioxyphenethylaminefor tyramine, the compounds Nhphe-Nbiph-Nhphe, Nzhphe-Nbiph-Nhphe andNoco-Nbiph-Nhphe were prepared. The compound Nhphe-Nbiph-Nhphe wasadditionally N-benzylated to produce Bz-Nhphe-Nbiph-Nhphe.

EXAMPLE 18

Proceeding as described in Examples 18 and 22 above, but substituting3-trifluoromethylphenethylamine, 2-chlorophenethylamine,3-chlorophenethylamine, 4-chlorophenethylamine,2,4-dichlorophenethylamine, 3-bromophenethylamine, 4-iodophenethylamine,3-hydroxyphenethylamine, 4-hydroxyphenethylamine,2,4-dihydroxyphenethylamine, 2-methylphenethylamine,3-methylphenethylamine, 4-methylphenethylamine,2,4-dimethylphenethylamine, 2,4,6-trimethylphenethylamine,3-ethylphenethylamine, 4-ethylphenethylamine, 4-hexylphenethylamine,3-nitrophenethylamine, 2-aminophenethylamine, 4-aminophenethylamine,2,4-diaminophenethylamine, 2-methoxyphenethylamine,3-methoxyphenethylamine, 4-methoxyphenethylamine,2,4-dimethoxyphenethylamine, 2,4,6-trimethoxyphenethylamine,3,4-dimethoxyphenethylamine, 2-ethoxyphenethylamine,3-ethoxyphenethylamine, 4-ethoxyphenethylamine, 3-propoxyphenethylamine,4-butoxyphenethylamine, 4-t-butoxyphenethylamine,3-methoxymethylphenethylamine, 4-methoxymethylphenethylamine,3-(2-methoxyethyl)phenethylamine, 4-(2-methoxyethyl)phenethylamine,4-(2-hydroxyethyl)phenethylamine, 4-(3-hydroxypropyl)phenethylamine,4-(2-hydroxyethoxy)phenethylamine, 4-phenylphenethylamine,4-(2-chlorophenyl)phenethylamine, 4-(2-aminophenyl)phenethylamine,3-(2,4,6-trimethylphenyl)phenethylamine, 4-phenoxyphenethylamine,4-(3-chlorophenoxy)phenethylamine, 4-(4-aminophenoxy)phenethylamine,3-benzylphenethylamine, 4-phenethylphenethylamine,3-acetylphenethylamine, 4-acetylphenethylamine,4-(2-phenoxyethyl)phenethylamine, and 3-benzyloxyphenethylamine forphenethylamine, and/or 3'-trifluoromethyl-4-aminobiphenyl,2'-chloro-4-aminobiphenyl, 3-chloro-4-aminobiphenyl,4'-chloro-4-aminobiphenyl, 2',4'-dichloro-4-aminobiphenyl,3-bromo-4-aminobiphenyl, 4'- iodo-4-aminobiphenyl,3'-hydroxy-4-aminobiphenyl, 4'-hydroxy-4-aminobiphenyl,2',4'-dihydroxy-4-aminobiphenyl, 2'-methyl-4-aminobiphenyl,3'-methyl-4-aminobiphenyl, 4'-methyl-4-aminobiphenyl,2',4'-dimethyl-4-aminobiphenyl, 2',4',6'-trimethyl-4-aminobiphenyl,2',3,4',5,6'-pentamethyl-4-aminobiphenyl, 3'-ethyl-4-aminobiphenyl,4'-ethyl-4-aminobiphenyl, 4'-hexyl-4-aminobiphenyl,3'-nitro-4-aminobiphenyl, 2'-amino-4-aminobiphenyl,4'-amino-4-aminobiphenyl, 2',4'-diamino-4-aminobiphenyl,2'-methoxy-4-aminobiphenyl, 3'-methoxy-4-aminobiphenyl,4'-methoxy-4-aminobiphenyl, 2',4'-dimethoxy-4-aminobiphenyl,2',4',6'-trimethoxy-4-aminobiphenyl, 3',4'-dimethoxy-4-aminobiphenyl,2'-ethoxy-4-aminobiphenyl, 3'-ethoxy-4-aminobiphenyl,4'-ethoxy-4-aminobiphenyl, 3'-propoxy-4-aminobiphenyl,4'-butoxy-4-aminobiphenyl, 4'-t-butoxy-4-aminobiphenyl,3'-methoxymethyl-4-aminobiphenyl, 4'-methoxymethyl-4-aminobiphenyl,3'-methoxyethyl-4-aminobiphenyl, 4'-methoxyethyl-4-aminobiphenyl,4'-hydroxyethyl-4-aminobiphenyl, 4'-hydroxypropyl-4-aminobiphenyl,4'-hydroxyethoxy-4-aminobiphenyl, 4'-phenyl-4-aminobiphenyl,4'-(2-chlorophenyl)-4-aminobiphenyl, 4'-(2-aminophenyl)-4-aminobiphenyl,3'-(2,4,6-trimethylphenyl)-4-aminobiphenyl, 4'-phenoxy-4-aminobiphenyl,4'-(3-chlorophenoxy)-4-aminobiphenyl,4'-(4-aminophenoxy)-4-aminobiphenyl, 3'-benzyl-4-aminobiphenyl,4'-phenethyl-4-aminobiphenyl, 3'-acetyl-4-aminobiphenyl,4'-acetyl-4-aminobiphenyl, 4'-(2-phenoxyethyl)-4-aminobiphenyl, and3'-benzyloxy-4-aminobiphenyl for 4-aminobiphenyl, and/or phenethylamine,3-trifluoromethylphenethylamine, 2-chlorophenethylamine,3-chlorophenethylaminechlorophenelchlorophenethylamine,2,6-dichlorophenethylamine, 3-bromophenethylamine,4-fluorophenethylamine, 3-hydroxyphenethylamine,2,5-dihydroxyphenethylamine, 2-methylphenethylamine,3-methylphenethylamine, 4-methylphenethylamine,2,4-dimethylphenethylamine, 2,4,6-trimethylphenethylamine,3-ethylphenethylamine, 4-ethylphenethylamine, 4-hexylphenethylamine,3-nitrophenethylamine, 2-aminophenethylamine, 4-aminophenethylamine,2,4-diaminophenethylamine, 2-methoxyphenethylamine,2,5-dimethoxyphenethylamine, 2,3-dimethoxyphenethylamine,3,5-dimethoxyphenethylamine, 3,4,5-trimethoxyphenethylamine,3-methoxyphenethylamine, 4-methoxyphenethylamine,2,4-dimethoxyphenethylamine, 2,4,6-trimethoxyphenethylamine,3,4-dimethoxyphenethylamine, 2-ethoxyphenethylamine,3-ethoxyphenethylamine, 4-ethoxyphenethylamine, 3-propoxyphenethylamine,4-butoxyphenethylamine, 4-t-butoxyphenethylamine,3-methoxymethylphenethylamine, 4-methoxymethylphenethylamine,3-methoxyethylphenethylamine, 4-methoxyethylphenethylamine,4-hydroxyethylphenethylamine, 4-hydroxypropylphenethylamine,4-hydroxyethoxyphenethylamine, 4-phenylphenethylamine,4-(2-chlorophenyl)phenethylamine, 4-(2-aminophenyl)phenethylamine,3-(2,4,6-trimethylphenyl)phenethylamine, 4-phenoxyphenethylamine,4-(3-chlorophenoxy)phenethylamine, 3,4-methylenedioxyphenethylamine,6-methoxy-3,4-methylenedioxyphenechylamine,2-methoxy-3,4-methylenedioxyphenethylamine,4,5-methylenedioxyphenechylamine,3-methoxy-4,5-methylenedioxyphenethylamine,4-(4-aminophenoxy)phenethylamine, 3-benzylphenethylamine,4-phenethylphenethylamine, 3-acetylphenethylamine,4-acetylphenethylamine, 4-(2-phenoxyethyl)phenethylamine, and3-benzyloxyphenethylamine for 4-hydroxyphenethylamine, the correspondingcompounds are prepared.

The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures can be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

We claim:
 1. A method for producing a poly(N-substituted)amide comprisedof different monomer units, said method comprising:(a) reacting a firstamine submonomer with a first acylating submonomer comprising a firstleaving group to a produce a first monomer unit comprising said firstleaving group, wherein said amine submonomer is bound to a substrate orto an acylated substrate; (b) reacting said first monomer unitcomprising said first leaving group with a second amine submonomer,whereby said second amine submonomer bonds to said first monomer unitthrough displacement of said first leaving group; (c) reacting saidsecond amine submonomer bonded to said first monomer with a secondacylating submonomer to produce a second monomer unit comprising asecond leaving group; and (d) optionally repeating steps (b) and (c)with additional amine and acylating submonomers to produce additionalmonomer units; wherein said method further comprises a washing stepbetween each of said reacting steps; whereby a poly(N-substituted)amideis produced.
 2. The method according to claim 1, wherein said substrateis not acylated.
 3. The method according to claim 2, wherein said firstamine submonomer is a secondary amine.
 4. The method according to claim1, wherein said first leaving group is a halogen.
 5. The methodaccording to claim 1, wherein said first acylating submonomer is ahaloacetic acid.
 6. The method according to claim 5, wherein saidholoacetic acid is selected from the group consisting of chloroaceticacid, bromoacetic and iodoacetic acid.
 7. The method according to claim1, wherein said substrate is acylated.
 8. The method according to claim7, wherein said acylated substrate comprises a substrate surface-boundacylating agent submonomer.
 9. The method according to claim 1, whereinsaid second amine submonomer is selected from the group consisting of aprimary amine, a secondary amine, an alkoxyamine, a semicarbazide, anacyl hydrazide and a carbazate.
 10. The method according to claim 9,wherein said second amine submonomer of step (b) is a primary amine. 11.The method according to claim 10, wherein said primary amine is selectedfrom the group consisting of 4-(2-aminoethyl)morpholine, aniline,benzylamine, cyclopentylamine, N-Boc-1,6-diaminohexane HCl, glycine-OtBuHCl, hexylamine, 2-methoxyethylamine, methylamine, and tyramine.
 12. Themethod according to claim 1, wherein steps (b) and (c) are repeated fromfive to twenty times.
 13. The method according to claim 1, wherein saidmonomer units of said poly(N-substituted)amide have the structuralformula: ##STR54## wherein: R is a sidechain;Z is a bond, --O--,--NC(O)W-- in which W-- is a bond, --O--, or --N--; and Y is ahydrocarbylene group or Ar, wherein Ar is selected from the groupconsisting of arylene, heteroarylene, cycloalkylene, cycloalkenylene,and heterocycloalkylene.
 14. A method for producing apoly(N-substituted)amide comprised of different monomer units, saidmethod comprising:(a) acylating a first amine submonomer bound to asubstrate with a first acylating submonomer comprising a first leavinggroup to a produce a first monomer unit comprising said first leavinggroup; (b) reacting said first monomer unit comprising said firstleaving group with a second amine submonomer, whereby said second aminesubmonomer bonds to said first monomer unit through displacement of saidfirst leaving group; (c) reacting said second amine submonomer bonded tosaid first monomer with a second acylating submonomer to produce asecond monomer unit comprising a second leaving group; and (d)optionally repeating steps (b) and (c) five to twenty times withadditional amine and acylating submonomers to produce five to twentyadditional monomer units; wherein said method further comprises awashing step between each of said reacting steps; whereby apoly(N-substituted)amide is produced.
 15. The method according to claim14, wherein said first amine submonomer is a secondary amine.
 16. Themethod according to claim 15, wherein said second amine submonomer isselected from the group consisting of a primary amine, a secondaryamine, an alkoxyamine, a semicarbazide, and acyl hydrazide and acarbazate.
 17. The method according to claim 16, wherein said secondamine submonomer is a primary amine.
 18. The method according to claim17, wherein said primary amine is selected from the group consisting of4-(2-aminoethyl)morpholine, aniline, benzylamine, cyclopentylamine,N-Boc-1,6-diaminohexane HCl, glycine-OtBu HCl, hexylamine,2-methoxyethylamine, methylamine, and tyramine.
 19. The method accordingto claim 14, wherein said first leaving group is a halogen.
 20. Themethod according to claim 14, wherein said first acylating submonomer isa haloacetic acid.
 21. The method according to claim 20, wherein saidhaloacetic acid is selected from the group consisting of chloroaceticacid, bromoacetic acid and iodoacetic acid.
 22. The method according toclaim 14, wherein said monomer units of said poly(N-substituted)amidehave the structural formula: ##STR55## wherein: R is a sidechain;Z is abond, --O--, --NC(O)W-- in which W-- is a bond, --O--, or --N--; and Yis a hydrocarbylene group or Ar, wherein Ar is selected from the groupconsisting of arylene, heteroarylene, cycloalkylene, cycloalkenylene,and heterocycloalkylene.
 23. A method for producing apoly(N-substituted)amide comprised of different monomer units, saidmethod comprising:(a) reacting an acylated substrate with a first aminesubmonomer; (b) reacting said first amine submonomer of step (a) with afirst acylating submonomer comprising a first leaving group to a producea first monomer unit comprising said first leaving group; (c) reactingsaid first monomer unit comprising said first leaving group with asecond amine submonomer, whereby said second amine submonomer bonds tosaid first monomer unit through displacement of said first leavinggroup; (d) reacting said second amine submonomer bonded to said firstmonomer with a second acylating submonomer to produce a second monomerunit comprising a second leaving group; and (e) optionally repeatingsteps (c) and (d) five to twenty times with additional amine andacylating submonomers to produce from five to twenty additional monomerunits; wherein said method further comprises a washing step between eachof said reacting steps; whereby a poly(N-substituted)amide is produced.24. The method according to claim 23, wherein said second aminesubmonomer is selected from the group consisting of a primary amine, asecondary amine, an alkoxyamine, a semicarbazide, and acyl hydrazide anda carbazate.
 25. The method according to claim 24, wherein said secondamine submonomer is a primary amine.
 26. The method according to claim25, wherein said primary amine is selected from the group consisting of4-(2-aminoethyl)morpholine, aniline, benzylamine, cyclopentylamine,N-Boc-1,6-diaminohexane HCl, glycine-OtBu HCl, hexylamine,2-methoxyethylamine, methylamine, and tyramine.
 27. The method accordingto claim 24, wherein said first leaving group is a halogen.
 28. Themethod according to claim 24, wherein said first acylating submonomer ofstep is a haloacetic acid.
 29. The method according to claim 28, whereinsaid haloacetic acid is selected from the group consisting ofchloroacetic acid, bromoacetic acid and iodoacetic acid.
 30. The methodaccording to claim 23, wherein said monomer units of saidpoly(N-substituted)amide have the structural formula: ##STR56## wherein:R is a sidechain;Z is a bond, --O--, --NC(O)W-- in which W-- is a bond,--O--, or --N--; and Y is a hydrocarbylene group or Ar, wherein Ar isselected from the group consisting of arylene, heteroarylene,cycloalkylene, cycloalkenylene, and heterocycloalkylene.